Methods of treating acne

ABSTRACT

A method of treating acne in a human in need thereof comprising administering systemically to said human a tetracycline compound in an amount that is effective to treat acne but has substantially no antibiotic activity, without administering a bisphosphonate compound.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/281,916, filed Apr. 5, 2001, and U.S. ProvisionalApplication No. 60/325,489, filed Sep. 26, 2001, both of which areincorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Acne is a common disease characterized by various types oflesions. The areas affected typically are areas of the skin wheresebaceous glands are largest, most numerous, and most active. Thelesions associated with acne are usually categorized as eithernon-inflammatory or inflammatory.

[0003] Non-inflammatory lesions include comedones. Comedones appear intwo forms, open and closed. Comedones are thought to arise from abnormalfollicular differentiation. Instead of undergoing shedding and dischargethrough the follicular orifice, abnormal desquamated cells(keratinocytes) become unusually cohesive, forming a microcomedo or amicroscopic hyperkeratotic plug in the follicular canal. The progressiveaccumulation of these microcomedones lead to visible comedones.

[0004] In its mildest form, acne is a more or less superficial disordercharacterized by slight, spotty skin irritations. In such cases,ordinary skin hygiene is typically a satisfactory treatment. In the moreinflammatory types of acne, however, pustules; infected cysts; and inextreme cases, canalizing, inflamed and infected sacs appear. Withouteffective treatment, these lesions may become extensive and leavepermanent, disfiguring scars.

[0005] Microorganisms, especially Propionibacterium acnes, are stronglyimplicated in the pathogenesis of acne. The microorganisms are thoughtto release microbial mediators of inflammation into the dermis ortrigger the release of cytokines from ductal keratinocytes.

[0006] Accordingly, the efficacy of antibiotics in treating acne isthought to be due, in significant part, to the direct inhibitory effectof the antibiotics on the growth and metabolism of these microorganisms.Systemically-administered tetracycline antibiotics, especiallyminocycline hydrochloride, are particularly effective in treating acne.

[0007] The tetracyclines are a class of compounds of which tetracyclineis the parent compound. Tetracycline has the following generalstructure:

[0008] The numbering system of the multiple ring nucleus is as follows:

[0009] Tetracycline, as well as the 5-hydroxy (oxytetracycline, e.g.Terramycin) and 7-chloro (chlorotetracycline, e.g. Aureomycin)derivatives, exist in nature, and are all well known antibiotics.Semisynthetic derivatives such as 7-dimethylaminotetracycline(minocycline) and 6α-deoxy-5-hydroxytetracycline (doxycycline) are alsoknown tetracycline antibiotics. Natural tetracyclines may be modifiedwithout losing their antibiotic properties, although certain elements ofthe structure must be retained to do so.

[0010] In addition to the direct antibiotic activity of tetracyclines,further activities of antibiotic tetracyclines have been investigatedfor possible therapeutic effects on acne.

[0011] For example, a study by Elewski et al., J. Amer. Acad. Dermatol.,8:807-812 (1983) suggests that acne therapy, consisting oforally-administered tetracycline at a total daily dose of 1000 mg, mayhave therapeutic anti-inflammatory effects in addition to antibioticeffects. In particular, it was found that the anti-inflammatory effectof tetracycline was, at least in part, due to inhibition of neutrophilchemotaxis induced by bacterial chemotactic factors.

[0012] A more recent study, performed by Eady et al., J. Invest.Dermatol., 101:86-91 (1993), evaluated the effects of oral minocyclineor tetracycline therapy on the cytokine and microflora content of opencomedones in acne patients. The total daily dose of minocyclineadministered was 100 mg. The total daily dose of tetracyclineadministered was 1000 mg.

[0013] Eady et al. found that the therapies upregulated the productionof bioactive IL-1α-like material and immunochemical IL-1β. IL-1 isconsidered to be a pro-inflammatory cytokine.

[0014] Accordingly to Eady et al., no overall decrease in the numbers ofpropionibacteria/mg of comedonal material was found. It is important tonote, however, that the numbers of propionibacteria/mg of comedonalmaterial are not expected to decrease in response to antibiotic therapy.Since the bacteria within comedones are encapsulated by the follicle,they are not susceptible to antibiotic treatment.

[0015] Another possible activity of tetracyclines in acne therapy wasinvestigated by Bodokh, I., et al., Acta. Derm. Venerol., 77:255-259(1997). Their study was designed to evaluate the action of minocyclineon sebaceous excretion in acne patients. A 100 mg daily dose ofminocycline was administered. A subclinical increase in seborrhoea wasreported. The authors propose that minocycline induces an increase inseborrhoea via a reduction in ductal obstruction. The mechanism by whichthe ductal obstruction is reduced is proposed to be a reduction inductal irritation. The authors suggest that the reduction of ductalirritation is due to minocycline's direct effect on P. acnes, orminocycline's effect on the lipase produced by P. acnes.

[0016] Bodokh et al. also found that during treatment no correlationexists between seborrhoea intensity and clinical severity of acne. Theauthors state that the lack of correlation shows that seborrhoea ispathogenic because it is the “culture medium” of P. acnes. Thus, it canbe concluded that the authors consider the antibiotic activity ofminocycline to be therapeutically significant with respect to acne.

[0017] Similarly, in a recent clinical study it was reported thattetracycline in sub-antibiotic doses had no clinical effect on acne.(Cunliffe et al., J. Am. Acad. Dermatol., 16:591-9 (1987).) Inparticular, a 100 mg total daily dose of minocycline and a 1.0 g totaldaily dose of tetracycline were found to be necessary to successfullytreat acne.

[0018] The antibiotic effects of antibiotics are generally directlyproportional to the dose administered of the antibiotics. Accordingly,in moderate to severe (i.e. inflammatory) forms of acne, oralantibiotics are typically administered at high doses. For example, inconventional acne therapy, tetracycline is administered at an initialdose of 500 to 2,000 mg/day, followed by a maintenance dose of 250-500mg/day.

[0019] Clearly, the state-of-the-art teaching is that the clinicalefficacy of systemically-administered tetracyclines in the treatment ofacne is due, at least in significant part, to the antibiotic effects ofthe tetracyclines. In addition to their antibiotic effects, it has beenproposed that tetracyclines reduce the number of inflammatory lesions(papules, pustules and nodules) by a variety of non-antibioticmechanisms. Such mechanisms include interfering with the chemotaxis ofpolymorphonuclear leukocytes (PMN) into the inflammatory lesion,inhibition of PMN derived collagenase, and by scavenging reactiveoxidative species produced by resident inflammatory cells.

[0020] There is no disclosure in the prior art of using either asub-antibiotic dose of an antibiotic tetracycline compound, or of usinga non-antibiotic tetracycline compound for the treatment of acne.

[0021] The use of tetracycline antibiotics, however, can lead toundesirable side effects. For example, the long term administration ofantibiotic tetracyclines can reduce or eliminate healthy microbialflora, such as intestinal flora, and can lead to the production ofantibiotic resistant organisms or the overgrowth of yeast and fungi.

[0022] Accordingly, there is a need for an effective treatment of acnewhich causes fewer undesirable side effects produced by thesystemically-administered antibiotics used in conventional acne therapy.

SUMMARY OF INVENTION

[0023] The present invention provides a method of treating acne in ahuman in need thereof. The method comprises administering systemicallyto the human a tetracycline compound in an amount that is effective totreat acne but has substantially no antibiotic activity (i.e.substantially no antimicrobial activity), without administering abisphosphonate compound.

[0024] Additionally, the present invention provides methods for reducingthe number of comedones, inhibiting oxidation of melanin, and/orinhibiting lipid-associated abnormal follicular differentiation in ahuman in need thereof. These methods comprise administering systemicallyto the human a tetracycline compound in an amount that is effective forits purpose, e.g., to reduce the number of comedones, to inhibitoxidation of melanin, and/or to inhibit lipid-associated abnormalfollicular differentiation, but has substantially no antibioticactivity.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 shows the photoirritancy factor (PIF) for some tetracyclinecompounds. For structure K, the compounds indicated are as follows: COLR7 R8 R9 308 hydrogen hydrogen amino 311 hydrogen hydrogen palmitamide306 hydrogen hydrogen dimethylamino

[0026] For structures L, M, N or O the compounds indicated are asfollows: COL R7 R8 R9 801 hydrogen hydrogen acetamido 802 hydrogenhydrogen dimethylaminoacetamido 804 hydrogen hydrogen nitro 805 hydrogenhydrogen amino

[0027] For structure P, R8 is hydrogen and R9 is nitro.

DETAILED DESCRIPTION

[0028] The present invention provides methods of treating acne. As usedherein, the term “acne” is a disorder of the skin characterized bypapules, pustules, cysts, nodules, comedones, and other blemishes orskin lesions. These blemishes and lesions are often accompanied byinflammation of the skin glands and pilosebaceous follicles, as well as,microbial, especially bacterial, infection.

[0029] For the purposes of this specification, acne includes all knowntypes of acne. Some types of acne include, for example, acne vulgaris,cystic acne, acne atrophica, bromide acne, chlorine acne, acneconglobata, acne cosmetica, acne detergicans, epidemic acne, acneestivalis, acne fulminans, halogen acne, acne indurata, iodide acne,acne keloid, acne mechanica, acne papulosa, pomade acne, premenstralacne, acne pustulosa, acne scorbutica, acne scrofulosorum, acneurticata, acne varioliformis, acne venenata, propionic acne, acneexcoriee, gram negative acne, steroid acne, nodulocystic acne and acnerosacea. Acne rosacea is characterized by inflammatory lesions(erythema) and permanent dilation of blood vessels (telangectasia).

[0030] The present invention is particularly effective in treatingcomedones, e.g., reducing the number of comedones. Both open and closedcomedones can be treated in accordance with the methods of thisinvention.

[0031] The present invention can also be used to treat certain othertypes of acneiform dermal disorders, e.g. perioral dermatitis,seborrheic dermatitis in the presence of acne, gram negativefolliculitis, sebaceous gland dysfunction, hiddradenitis suppurativa,pseudo-folliculitis barbae, or folliculitis.

[0032] The method comprises the administration of a tetracyclinecompound to a human in an amount which is effective for its purposee.g., the treatment of acne, including reducing the number of comedones,but which has substantially no antibiotic activity.

[0033] The tetracycline compound can be an antibiotic or non-antibioticcompound. The tetracycline compound has the general tetracyclinestructure indicated above, or a derivative thereof.

[0034] Some examples of antibiotic (i.e. antimicrobial) tetracyclinecompounds include doxycycline, minocycline, tetracycline,oxytetracycline, chlortetracycline, demeclocycline, lymecycline andtheir pharmaceutically acceptable salts. Doxycycline is preferablyadministered as its hyclate salt or as a hydrate, preferablymonohydrate.

[0035] Non-antibiotic tetracycline compounds are structurally related tothe antibiotic tetracyclines, but have had their antibiotic activitysubstantially or completely eliminated by chemical modification. Forexample, non-antibiotic tetracycline compounds are capable of achievingantibiotic activity comparable to that of tetracycline or doxycycline atconcentrations at least about ten times, preferably at least abouttwenty five times, greater than that of tetracycline or doxycycline,respectively.

[0036] Examples of chemically modified non-antibiotic tetracyclines(CMTs) include 4-de(dimethylamino)tetracycline (CMT-1),tetracyclinonitrile (CMT-2),6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),7-chloro-4-de(dimethylamino)tetracycline (CMT-4), tetracycline pyrazole(CMT-5), 4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),4-de(dimethylamino-12α-deoxytetracycline (CMT-7),6-deoxy-5α-hydroxy-4-de(dimethylamino)tetracycline (CMT-8),4-de(dimethylamino)-12α-deoxyanhydrotetracycline (CMT-9),4-de(dimethylamino)minocycline (CMT-10).

[0037] Further examples of chemically modified non-antibiotictetracyclines include Structures C-Z. (See Index of Structures.)

[0038] Tetracycline derivatives, for purposes of the invention, may beany tetracycline derivative, including those compounds disclosedgenerically or specifically in co-pending U.S. patent application Ser.No. 09/573,654 filed on May 18, 2000, which are herein incorporated byreference.

[0039] The minimal amount of the tetracycline compound administered to ahuman is the lowest amount capable of providing effective treatment ofacne. Effective treatment is a reduction or inhibition of the blemishesand lesions associated with acne. The amount of the tetracyclinecompound is such that it does not significantly prevent the growth ofmicrobes, e.g. bacteria.

[0040] Two ways in which to describe the administered amount of atetracycline compound is by daily dose, and by serum level.

[0041] For example, tetracycline compounds that have significantantibiotic activity may be administered in a dose (i.e. amount) which is10-80% of the antibiotic dose. More preferably, the antibiotictetracycline compound is administered in a dose which is 40-70% of theantibiotic dose.

[0042] Some examples of antibiotic doses of members of the tetracyclinefamily include 50, 75, and 100 mg/day of doxycycline; 50, 75, 100, and200 mg/day of minocycline; 250 mg of tetracycline one, two, three, orfour times a day; 1000 mg/day of oxytetracycline; 600 mg/day ofdemeclocycline; and 600 mg/day of lymecycline.

[0043] Examples of the maximum non-antibiotic doses of tetracyclinesbased on steady-state pharmacokinetics are as follows: 20 mg/twice a dayfor doxycycline; 38 mg of minocycline one, two, three or four times aday; and 60 mg of tetracycline one, two, three or four times a day.

[0044] In a preferred embodiment, to reduce the number of comedones,doxycycline is administered in a daily amount of from about 30 to about60 milligrams, but maintains a concentration in human plasma below thethreshold for a significant antibiotic effect.

[0045] In an especially preferred embodiment, doxycycline hyclate isadministered at a 20 milligram dose twice daily. Such a formulation issold for the treatment of periodontal disease by CollaGenexPharmaceuticals, Inc. of Newtown, Pa. under the trademark Periostat®.

[0046] Example 38 below summarizes a clinical study using 20 mgdoxycycline hyclate tablets administered twice a day. A significantreduction in the number of comedones was observed. This reduction in thenumber of comedones is unexpected. The reduction is particularlyunexpected since, as can be seen from the microbiology results inExample 38, the treatment with doxycycline resulted in no reduction ofskin microflora vis-à-vis a placebo control.

[0047] The administered amount of a tetracycline compound described byserum levels follows.

[0048] An antibiotic tetracycline compound is advantageouslyadministered in an amount that results in a serum tetracyclineconcentration which is 10-80% of the minimum antibiotic serumconcentration. The minimum antibiotic serum concentration is the lowestconcentration known to exert a significant antibiotic effect.

[0049] Some examples of the approximate antibiotic serum concentrationsof members of the tetracycline family follow.

[0050] For example, a single dose of two 100 mg minocycline HCl tabletsadministered to adult humans results in minocycline serum levels rangingfrom 0.74 to 4.45 μg/ml over a period of an hour. The average level is2.24 μg/ml.

[0051] Two hundred and fifty milligrams of tetracycline HCl administeredevery six hours over a twenty-four hour period produces a peak plasmaconcentration of approximately 3 μg/ml. Five hundred milligrams oftetracycline HCl administered every six hours over a twenty-four hourperiod produces a serum concentration level of 4 to 5 μg/ml.

[0052] In one embodiment, the tetracycline compound can be administeredin an amount which results in a serum concentration between about 0.1and 10.0 μg/ml, more preferably between 0.3 and 5.0 μg/ml. For example,doxycycline is administered in an amount which results in a serumconcentration between about 0.1 and 0.8 μg/ml, more preferably between0.4 and 0.7 μg/ml.

[0053] Some examples of the plasma antibiotic threshold levels oftetracyclines based on steady-state pharmacokinetics are as follows: 1.0μg/ml for doxycycline; 0.8 μg/ml for minocycline; and 0.5 μg/ml fortetracycline.

[0054] Non-antibiotic tetracycline compounds can be used in higheramounts than antibiotic tetracyclines, while avoiding the indiscriminatekilling of microbes, and the emergence of resistant microbes. Forexample, 6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3) maybe administered in doses of about 40 to about 200 mg/day, or in amountsthat result in serum levels of about 1.55 μg/ml to about 10 μg/ml.

[0055] The actual preferred amounts of tetracycline compounds in aspecified case will vary according to the particular compositionsformulated, the mode of application, the particular sites ofapplication, and the subject being treated.

[0056] The tetracycline compounds can be in the form of pharmaceuticallyacceptable salts of the compounds. The term “pharmaceutically acceptablesalt” refers to a salt prepared from tetracycline compounds andpharmaceutically acceptable non-toxic acids or bases. The acids may beinorganic or organic acids of tetracycline compounds. Examples ofinorganic acids include hydrochloric, hydrobromic, hydroiodic, sulfuric,and phosphoric acids. Examples of organic acids include carboxylic andsulfonic acids. The radical of the organic acids may be aliphatic oraromatic. Some examples of organic acids include formic, acetic,phenylacetic, propionic, succinic, glycolic, glucuronic, maleic, furoic,glutamic, benzoic, anthranilic, salicylic, phenylacetic, mandelic,embonic (pamoic), methanesulfonic, ethanesulfonic, panthenoic,benzenesulfonic, stearic, sulfanilic, alginic, tartaric, citric,gluconic, gulonic, arylsulfonic, and galacturonic acids. Appropriateorganic bases may be selected, for example, fromN,N-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine), and procaine.

[0057] The tetracycline compounds mentioned above, especiallydoxycycline and minocycline, are unexpectedly effective in reducing thenumber of comedones when administered at a dose which has substantiallyno antibiotic effect. Preferably the reduction is at least about 20%greater than for a placebo control, more preferably at least about 30%greater than for a placebo control, most preferably at least about 40%greater than for a placebo control, and optimally at least about 50%greater than for a placebo control.

[0058] The inventors are not certain of, and do not wish to be limitedby, any particular mechanism of action. Nevertheless, it is believedthat the ability of tetracyclines, such as doxycycline, to inhibitoxidation of melanin and to inhibit lipid-associated abnormal folliculardifferentiation prevents keratinocytes from becoming cohesive, therebyinhibiting the formation of comedones.

[0059] Preferably, the tetracycline compounds have low phototoxicity, orare administered in an amount that results in a serum level at which thephototoxicity is acceptable. Phototoxicity is a chemically-inducedphotosensitivity. Such photosensitivity renders skin susceptible todamage, e.g. sunburn, blisters, accelerated aging, erythemas andeczematoid lesions, upon exposure to light, in particular ultravioletlight. The preferred amount of the tetracycline compound produces nomore phototoxicity than is produced by the administration of a 40 mgtotal daily dose of doxycycline.

[0060] Phototoxicity can be evaluated in terms of a photoirritancyfactor (PIF), as described in the examples. A PIF value of about 1.0indicates that a compound is considered to have no measurablephototoxicity.

[0061] The low phototoxic derivatives preferably have PIF values nogreater than about 5, preferably no greater than about 2, morepreferably no greater than about 1.5, most preferably no greater thanabout 1.2, and optimally about 1.

[0062] Some antibiotic tetracyclines having low phototoxicity include,for example, minocycline and tetracyline.

[0063] Some non-antibiotic tetracyclines having low phototoxicityinclude, but are not limited to, tetracycline compounds having thegeneral formulae:

Structure K

[0064] wherein: R7, R8, and R9 taken together in each case, have thefollowing meanings: R7 R8 R9 hydrogen hydrogen amino hydrogen hydrogenpalmitamide hydrogen hydrogen dimethylamino and STRUCTURE L STRUCTURE MSTRUCTURE N STRUCTURE O

[0065] wherein: R7, R8, and R9 taken together in each case, have thefollowing meanings: R7 R8 R9 hydrogen hydrogen acetamido hydrogenhydrogen dimethylaminoacetamido hydrogen hydrogen nitro hydrogenhydrogen amino and STRUCTURE P

[0066] wherein: R8, and R9 taken together are, respectively, hydrogenand nitro.

[0067] The tetracycline compounds are administered without administeringa bisphosphonate compound. Bisphosphonates compounds are related toinorganic pyrophosphonic acid. The bisphosphonates include, asnon-limiting examples, alendronate((4-amino-1-hydroxybutylidene)bisphosphonic acid), clodronate(dichloromethane diphosphonic acid), etidronate ((1-hydroxyethylidene)diphosphanic acid) and pamidronate ((3-amino-1-hydroxypropylidene)bisphosphonic acid); also risedronate([-hydroxy-2-(3-pyridinyl)ethylidene]bisphosphonic acid), tiludronate,i.e., tiludronic acid ([(4-chlorophenyl)thio]methylene]bisphosphonicacid) and zolendronate.

[0068] The tetracycline compounds may, for example, be administeredsystemically. For the purposes of this specification, “systemicadministration” means administration to a human by a method that causesthe compounds to be absorbed into the bloodstream.

[0069] For example, the tetracyclines compounds can be administeredorally by any method known in the art. For example, oral administrationcan be by tablets, capsules, pills, troches, elixirs, suspensions,syrups, wafers, chewing gum and the like.

[0070] Additionally, the tetracycline compounds can be administeredenterally or parenterally, e.g., intravenously; intramuscularly;subcutaneously, as injectable solutions or suspensions;intraperitoneally; or rectally. Administration can also be intranasally,in the form of, for example, an intranasal spray; or transdermally, inthe form of, for example, a patch.

[0071] For the pharmaceutical purposes described above, the tetracyclinecompounds of the invention can be formulated per se in pharmaceuticalpreparations optionally with a suitable pharmaceutical carrier (vehicle)or excipient as understood by practitioners in the art. Thesepreparations can be made according to conventional chemical methods.

[0072] In the case of tablets for oral use, carriers which are commonlyused include lactose and corn starch, and lubricating agents such asmagnesium stearate are commonly added. For oral administration incapsule form, useful carriers include lactose and corn starch. Furtherexamples of carriers and excipients include milk, sugar, certain typesof clay, gelatin, stearic acid or salts thereof, calcium stearate, talc,vegetable fats or oils, gums and glycols.

[0073] When aqueous suspensions are used for oral administration,emulsifying and/or suspending agents are commonly added. In addition,sweetening and/or flavoring agents may be added to the oralcompositions.

[0074] For intramuscular, intraperitoneal, subcutaneous and intravenoususe, sterile solutions of the tetracycline compounds can be employed,and the pH of the solutions can be suitably adjusted and buffered. Forintravenous use, the total concentration of the solute(s) can becontrolled in order to render the preparation isotonic.

[0075] The tetracycline compounds of the present invention can furthercomprise one or more pharmaceutically acceptable additionalingredient(s) such as alum, stabilizers, buffers, coloring agents,flavoring agents, and the like.

[0076] The tetracycline compound may be administered intermittently. Forexample, the tetracycline compound may be administered 1-6 times a day,preferably 1-4 times a day.

[0077] Alternatively, the tetracycline compound may be administered bysustained release. Sustained release administration is a method of drugdelivery to achieve a certain level of the drug over a particular periodof time. The level typically is measured by serum concentration. Furtherdescription of methods of delivering tetracycline compounds by sustainedrelease can be found in the patent application, “Controlled Delivery ofTetracycline and Tetracycline Derivatives,” filed on Apr. 5, 2001 andassigned to CollaGenex Pharmaceuticals, Inc. of Newtown, Pa. Theaforementioned application is incorporated herein by reference in itsentirety. For example, 40 milligrams of doxycycline may be administeredby sustained release over a 24 hour period.

[0078] In the embodiment in which the tetracycline compound is anon-antibiotic tetracycline compound, administration can include topicalapplication. Particular non-antibiotic tetracycline compounds have onlylimited biodistribution, e.g. CMT-5. In such cases, topical applicationis the preferred method of administration of the compound.

[0079] Carrier compositions deemed to be suited for topical use includegels, salves, lotions, creams, ointments and the like. Thenon-antibiotic tetracycline compound can also be incorporated with asupport base or matrix or the like which can be directly applied toskin.

[0080] Topical application of non-antibiotic tetracycline compounds areeffective in treating acne while not inducing significant toxicity inthe human. For example, amounts of up to about 25% (w/w) in a vehicleare effective. Amounts of from about 0.1% to about 10% are preferred.

[0081] Combined or coordinated topical and systemic administration ofthe tetracycline compounds is also contemplated under the invention. Forexample, a non-absorbable non-antibiotic tetracycline compound can beadministered topically, while a tetracycline compound capable ofsubstantial absorption and effective systemic distribution in a humancan be administered systemically.

[0082] The tetracycline compounds are prepared by methods known in theart. For example, natural tetracyclines may be modified without losingtheir antibiotic properties, although certain elements of the structuremust be retained. The modifications that may and may not be made to thebasic tetracycline structure have been reviewed by Mitscher in TheChemistry of Tetracyclines, Chapter 6, Marcel Dekker, Publishers, NewYork (1978). According to Mitscher, the substituents at positions 5-9 ofthe tetracycline ring system may be modified without the complete lossof antibiotic properties. Changes to the basic ring system orreplacement of the substituents at positions 1-4 and 10-12, however,generally lead to synthetic tetracyclines with substantially less oreffectively no antibiotic activity.

[0083] Further methods of preparing the tetracycline compounds aredescribed in the examples.

EXAMPLES

[0084] The following examples serve to provide further appreciation ofthe invention but are not meant in any way to restrict the effectivescope of the invention.

[0085] Preparation of Compounds

Example 14-Dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-nitrotetracyclineSulfate

[0086] To a solution of one millimole of4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracycline in 25ml of concentrated sulfuric acid at 0° C. was added 1.05 mmole ofpotassium nitrate. The resulting solution was stirred at ice bathtemperature for 15 minutes and poured in one liter of cold ether withstirring. The precipitated solid was allowed to settle and the majorityof solvent decanted. The remaining material was filtered through asintered glass funnel and the collected solid was washed well with coldether. The product was dried in a vacuum desiccator overnight.

Example 29-amino-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclineSulfate

[0087] To a solution of 300 mg of the 9-nitro compound from example 1,in 30 ml of ethanol was added 50 mg of PtO₂. The mixture washydrogenated at atmospheric pressure until the theoretical amount ofhydrogen was absorbed. The system is flushed with nitrogen, the catalystPtO₂ is filtered and the filtrate added dropwise to 300 ml of ether. Theproduct that separates is filtered and dried in a vacuum desiccator.

Example 39-Acetamido-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclineSulfate

[0088] To a well stirred cold solution of 500 mg of9-amino-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclinesulfate from example 2, in 2.0 ml of 1.3-dimethyl-2-imidazolidinone, 500mg of sodium bicarbonate was added followed by 0.21 ml of acetylchloride. The mixture is stirred at room temperature for 30 minutes,filtered and the filtrate was added dropwise to 500 ml of ether. Theproduct that separated was filtered and dried in a vacuum desiccator.

Example 44-Dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-diazoniumtetracyclineSulfate

[0089] To a solution of 0.5 g of9-amino-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclinesulfate, from example 2, in 10 ml of 0.1N hydrochloric acid in methanolcooled in an ice bath, 0.5 ml of n-butyl nitrite was added. The solutionwas stirred at ice bath temperature for 30 minutes and then poured into250 ml of ether. The product that separated was filtered, washed withether and dried in a vacuum desiccator.

Example 59-Azido-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclineSulfate

[0090] To a solution of 0.3 mmole of4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-diazoniumtetracyclinesulfate, from example 4, 10 ml of 0.1 N methanolic hydrogen chloride wasadded 0.33 mmole of sodium azide. The mixture was stirred at roomtemperature for 1.5 hours. The reaction mixture was then poured into 200ml of ether. The product that separated was filtered and dried in avacuum desiccator.

Example 69-Amino-8-chloro-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclineSulfate

[0091] One gram of9-azido-4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxytetracyclinehydrochloride, from example 4, was dissolved in 10 ml of concentratedsulfuric acid saturated with HCL at 0° C. The mixture was stirred at icebath temperature for 1.5 hours and then slowly added dropwise to 500 mlof cold ether. The product that separated was filtered, washed withether and dried in a vacuum desiccator.

Example 74-Dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-ethoxythiocarbonylthiotetracyclineSulfate

[0092] A solution of 1.0 mmole of4-dedimethylamino-7-dimethylamino-6-demethyl-6-deoxy-9-diazoniumtetracyclinesulfate, from example 4, in 15 ml of water was added to a solution of1.15 mmole of potassium ethyl xanthate in 15 ml of water. The mixturewas stirred at room temperature for one hour. The product separated andwas filtered and dried in a vacuum desiccator.

Example 8A General Procedure for Nitration

[0093] To 1 mmole of a 4-dedimethylamino-6-deoxytetracycline in 25 ml ofconcentrated sulfuric acid at 0° C. was added 1 mmole of potassiumnitrate with stirring. The reaction solution was stirred for 15 minutesand then poured into 100 g of chopped ice. The aqueous solution wasextracted 5 times with 20 ml of butanol each time. The butanol extractswere washed three times with 10 ml of water each time, and concentratedin vacuo to a volume of 25 ml. The light yellow crystalline solid whichprecipitated was filtered, washed with 2 ml of butanol and dried invacuo at 60° C. for 2 hours. This solid was a mixture of the twomononitro isomers.

Example 8B 4-Dedimethylamino-6-deoxy-9-nitrotetracycline

[0094] To 980 mg of the nitration product from4-dedimethylamino-6-deoxytetracycline (a mixture of the 2 isomers) in 25ml of methanol was added enough triethylamine to dissolve the solid. Thefiltered solution (pH 9.0) was adjusted to pH 5.2 with concentratedsulfuric acid. A crystalline yellow solid (236 mg.) was obtained (29%yield). The material at this point was quite pure and contained onlysmall amounts of the 7-isomer. Final purification was accomplished byliquid partition chromatography using a diatomaceous earth packed columnand the solvent system: chloroform: butanol: 0.5 M phosphate buffer (pH2) (16:1:10).

Example 9 4-Dedimethylamino-6-deoxy-7-nitrotetracycline

[0095] The methanol filtrate from example 8 was immediately adjusted topH 1.0 with concentrated sulfuric acid. The light yellow crystallinesolid, which was obtained as the sulfate salt. A purified free base wasobtained by adjusting an aqueous solution of the sulfate salt (25 mg/ml)to pH 5.2 with 2 N sodium carbonate.

Example 10 9-Amino-4-dedimethylamino-6-deoxytetracycline

[0096] To a solution of 300 mg of the 9-nitro compound, prepared inexample 8, in 30 ml of ethanol was added 50 mg of PtO₂. The mixture washydrogenated at atmospheric pressure until the theoretical amount ofhydrogen was absorbed. The system is flushed with nitrogen, the PtO₂catalyst is filtered and the filtrate added dropwise to 300 ml of ether.The solid that separates is filtered and dried in a vacuum desiccator.

Example 11 9-Acetamido-4-dedimethylamino-6-deoxytetracycline Sulfate

[0097] To well stirred cold solution of 500 mg of9-amino-4-dedimethylamino-6-deoxytetracycline sulfate, from example 10,in 2.0 ml of 1,3-dimethyl-2-imidazolidinone was added 500 mg of sodiumbicarbonate followed by 0.21 ml of acetyl chloride. The mixture wasstirred at room temperature for 30 minutes, filtered and the filtratewas added dropwise to 500 ml of ether. The solid that separated wasfiltered and dried in a vacuum desiccator.

Example 12 4-Dedimethylamino-6-deoxy-9-diazoniumtetracycline Sulfate

[0098] To a solution of 0.5 g of9-amino-4-dedimethylamino-6-deoxytetracycline sulfate, from example 10,in 10 ml of 0.1N hydrochloric acid in methanol cooled in an ice bath wasadded 0.5 ml of n-butyl nitrite. The solution was stirred at ice bathtemperature for 30 minutes and the poured into 250 ml of ether. Thesolid that separated was filtered, washed with ether and dried in avacuum desiccator.

Example 13 9-Azido-4-dedimethylamino-6-deoxytetracycline Sulfate

[0099] To a solution of 0.3 mmole of4-dedimethylamino-6-deoxy-9-diazoniumtetracycline sulfate, of example12, 10 ml of 0.1 N methanolic hydrogen chloride was added 0.33 mmole ofsodium azide. The mixture was stirred at room temperature for 1.5 hours.The reaction mixture was then poured into 200 ml of ether. The solidthat separated was filtered and dried in a vacuum desiccator.

Example 14 9-Amino-8-chloro-4-dedimethylamino-6-deoxytetracyclineSulfate

[0100] One gram of9-azido-4-dedimethylamino-7-dimethylamino-6-deoxytetracyclinehydrochloride, from example 13, was dissolved in 10 ml of concentratedsulfuric acid saturated with HCL at 0° C. The mixture was stirred at icebath temperature for 1.5 hours and then slowly added dropwise to 500 mlof cold ether. The solid that separated was filtered, washed and etherand dried in a vacuum desiccator.

Example 154-Dedimethylamino-6-deoxy-9-ethoxythiocarbonylthiotetracycline Sulfate

[0101] A solution of 1.0 mmole of4-dedimethylamino-6-deoxy-9-diazoniumtetracycline sulfate, from example12, in 15 ml of water was added to a solution of 1.15 mmole of potassiumethyl xanthate in 15 ml of water. The mixture was stirred at roomtemperature for one hour. The solid that separated was filtered anddried in a vacuum desiccator.

Example 16 9-Dimethylamino-4-dedimethylamino-6-deoxytetracycline Sulfate

[0102] To a solution of 100 mg. of the 9-amino compound from example 10,in 10 ml of ethylene glycol monomethyl ether is added 0.05 ml ofconcentrated sulfuric acid, 0.4 ml. of a 40% aqueous formaldehydesolution and 100 mg of a 10% palladium on carbon catalyst. The mixtureis hydrogenated under atmospheric pressure and room temperature for 20minutes. The catalyst was filtered and the filtrate was evaporated todryness under reduced pressure. The residue is dissolved in 5 ml ofmethanol and this solution was added to 100 ml of ether. The productthat separated was filtered and dried, yield, 98 mg.

Example 17 7-Amino-4-dedimethylamino-6-deoxytetracycline

[0103] This compound can be made using Procedure A or B. Procedure A. Toa solution of 300 mg of the 7-nitro compound, from example 1, in 30 mlof ethanol was added 50 mg of PtO₂. The mixture was hydrogenated atatmospheric pressure until the theoretical amount of hydrogen wasabsorbed. The system is flushed with nitrogen, the catalyst PtO₂ isfiltered and the filtrate added dropwise to 300 ml of ether. The solidthat separates is filtered and dried in a vacuum desiccator.

[0104] Procedure B. 1 g of 6-deoxy-4-dedimethylamino-tetracycline wasdissolved in 7.6 ml THF and 10.4 ml methanesulfonic acid at −10° C.After warming the mixture to 0° C. a solution of 0.86 g of dibenzylazodicarboxylate was added and the mixture stirred for 2 hours at 0° C.to yield7-[1,2-bis(carbobenzyloxy)hydrazino]-4-dedimethylamino-6-deoxytetracycline.A solution of 1 millimole of this material in 70 ml 2-methoxyethanol,and 300 mg 10% Pd-C was hydrogenated at room temperature to give7-amino-6-deoxy-4-dedimethylaminotetracycline.

Example 18 7-Amino-6-deoxy-5-hydroxy-4-dedimethylaminotetracycline

[0105] 1 g of 6-deoxy-5-hydroxy-4-dedimethylaminotetracycline 3 wasdissolved in 7.6 ml THF and 10.4 ml methanesulfonic acid at −10° C.After warming the mixture to 0° C. a solution of 0.86 g dibenzylazodicarboxylate in 0.5 ml THF was added and the mixture stirred for 2hours at 0° C. to yield7-[1,2-bis(carbobenzyloxy)hydrazino]-4-dedimethylamino-6-deoxy-5-hydroxytetracycline.A solution of 1 millimole of this material in 70 ml 2-methoxyethanol,and 300 mg 10% Pd-C was hydrogenated at room temperature to give7-amino-6-deoxy-5-hydroxytetracycline.

Example 19 7-Acetamido-4-dedimethylamino-6-deoxy-5-hydroxytetracyclineSulfate

[0106] To well stirred cold solution of 500 mg of7-amino-4-dedimethylamino-6-deoxy-5-hydroxytetracycline sulfate, fromexample 18, in 2.0 ml of 1,3-dimethyl-2-imidazolidinone was added 500 mgof sodium bicarbonate followed by 0.21 ml of acetyl chloride. Themixture was stirred at room temperature for 30 minutes, filtered and thefiltrate was added dropwise to 500 ml of ether. The solid that separatedwas filtered and dried in a vacuum desiccator.

Example 20 4-Dedimethylamino-6-deoxy-5-hydroxy-7-diazoniumtetracyclineHydrochloride

[0107] To a solution of 0.5 g of7-amino-4-dedimethylamino-6-deoxy-5-hydroxytetracycline sulfate, fromexample 20, in 10 ml of 0.1N hydrochloric acid in methanol cooled in anice bath was added 0.5 ml of n-butyl nitrite. The solution was stirredat ice bath temperature for 30 minutes and then poured into 250 ml ofether. The solid that separated was filtered, washed with ether anddried in a vacuum desiccator.

Example 21 7-Azido-4-dedimethylamino-6-deoxy-5-hydroxytetracycline

[0108] To a solution of 0.3 mmole of4-dedimethylamino-6-deoxy-5-hydroxy-7-diazoniumtetracyclinehydrochloride, from example 20, 10 ml of 0.1 N methanolic hydrogenchloride was added 0.33 mmole of sodium azide. The mixture was stirredat room temperature for 1.5 hours. The reaction mixture was then pouredinto 200 ml of ether. The solid that separated was filtered and dried ina vacuum desiccator.

Example 227-Amino-8-chloro-4-dedimethylamino-6-deoxy-5-hydroxytetracycline Sulfate

[0109] One gram of7-azido-4-dedimethylamino-7-dimethylamino-6-deoxy-5-hydroxytetracyclinesulfate, from example 21, was dissolved in 10 ml of concentratedsulfuric acid (previously saturated with hydrogen chloride) at 0° C. Themixture was stirred at ice bath temperature for 1.5 hours and thenslowly added dropwise to 500 ml of cold ether. The solid that separatedwas filtered, washed with ether and dried in a vacuum desiccator.

Example 234-Dedimethylamino-6-deoxy-5-hydroxy-7-ethoxythiocarbonylthiotetracycline

[0110] A solution of 1.0 mmole of4-dedimethylamino-6-deoxy-5-hydroxy-7-diazoniumtetracyclinehydrochloride, from example 20, in 15 ml of water was added to asolution of 1.15 mmole of potassium ethyl xanthate in 15 ml of water.The mixture was stirred at room temperature for one hour. The solid thatseparated was filtered and dried in a vacuum desiccator.

Example 247-Dimethylamino-4-dedimethylamino-6-deoxy-5-hydroxytetracycline Sulfate

[0111] To a solution of 100 mg of the 7-amino compound in 10 ml ofethylene glycol monomethyl ether is added 0.05 ml of concentratedsulfuric acid, 0.4 ml of a 40% aqueous formaldehyde solution and 100 mgof a 10% palladium on carbon catalyst. The mixture is reduced withhydrogen at atmospheric pressure and room temperature for 20 minutes.The catalyst was filtered and the filtrate was evaporated to drynessunder reduced pressure. The residue is dissolved in 5 ml of methanol andthis solution was added to 100 ml of ether. The product that separatedwas filtered and dried, yield, 78 mg.

Example 25 7-Diethylamino-4-dedimethylamino-5-hydroxytetracyclineSulfate

[0112] To a solution of 100 mg of the 7-amino compound in 10 ml ofethylene glycol monomethyl ether is added 0.05 ml of concentratedsulfuric acid, 0.4 ml of acetaldehyde and 100 mg of a 10% palladium oncarbon catalyst. The mixture is reduced with hydrogen at atmosphericpressure at room temperature for 20 minutes. The catalyst was filteredand filtrate was evaporated to dryness under reduced pressure. Theresidue is dissolved in 5 ml of methanol and this solution was added to100 ml of ether. The product that separated was filtered and dried.

Example 26 4-Dedimethylamino-6-deoxy-7-diazoniumtetracyclineHydrochloride

[0113] To a solution of 0.5 g. of7-amino-4-dedimethylamino-6-deoxytetracycline sulfate, from example 17,in 10 ml of 0.1N hydrochloric acid in methanol cooled in an ice bath wasadded 0.5 ml of n-butyl nitrite. The solution was stirred at ice bathtemperature for 30 minutes and then poured into 250 ml of ether. Thesolid that separated was filtered, washed with ether and dried in avacuum desiccator.

Example 27 7-Azido-4-dedimethylamino-6-deoxytetracycline

[0114] To a solution of 0.3 mmole of4-dedimethylamino-6-deoxy-7-diazoniumtetracycline hydrochloride, fromexample 26, 10 ml of 0.1 N methanolic hydrogen chloride was added 0.33mmole of sodium azide. The mixture was stirred at room temperature for1.5 hours. The reaction mixture was then poured into 200 ml of ether.The solid that separated was filtered and dried in a vacuum desiccator.

Example 28 7-Amino-8-chloro-4-dedimethylamino-6-deoxytetracyclineSulfate

[0115] One gram of7-azido-4-dedimethylamino-7-dimethylamino-6-deoxytetracycline sulfatewas dissolved in 10 ml of concentrated sulfuric acid (previouslysaturated with hydrogen chloride) at 0° C. The mixture was stirred atice bath temperature for 1.5 hours and then slowly added dropwise to 500ml of cold ether. The solid that separated was filtered, washed withether and dried in a vacuum desiccator.

Example 294-Dedimethylamino-6-deoxy-7-ethoxythiocarbonylthiotetracycline

[0116] A solution of 1.0 mmole of4-dedimethylamino-6-deoxy-7-diazoniumtetracycline hydrochloride, fromexample 26, in 15 ml of water was added to a solution of 1.15 mmole ofpotassium ethyl xanthate in 15 ml of water. The mixture was stirred atroom temperature for one hour. The solid that separated was filtered anddried in a vacuum desiccator.

Example 30 7-Dimethylamino-4-dedimethylamino-6-deoxytetracycline Sulfate

[0117] To a solution of 100 mg of the 7-amino compound, from example 26,in 10 ml of ethylene glycol monomethyl ether is added 0.05 ml ofconcentrated sulfuric acid, 0.4 ml of a 40% aqueous formaldehydesolution and 100 mg of a 10% palladium on carbon catalyst. The mixtureis reduced with hydrogen at atmospheric pressure and room temperaturefor 20 minutes. The catalyst was filtered and the filtrate wasevaporated to dryness under reduced pressure. The residue is dissolvedin 5 ml of methanol and this solution was added to 100 ml of ether. Theproduct that separated was filtered and dried.

Example 319-Acetamido-8-chloro-4-dedimethylamino-7-dimethylamino-6-deoxy-6-demethyltetracycline

[0118] To well stirred cold solution of 500 mg of9-amino-8-chloro-4-dedimethylamino-6-deoxy-6-demethyl-7-dimethyl aminotetracycline sulfate, from example 6, in 2.0 ml of 1,3-dimethyl-2-imidazolidinone was added 500 mg of sodium bicarbonate followed by0.21 ml. of acetyl chloride. The mixture was stirred at room temperaturefor 30 minutes, filtered and the filtrate was added dropwise to 500 mlof ether. The solid that separated was filtered and dried in a vacuumdesiccator.

Example 328-Chloro-4-dedimethylamino-7-dimethylamino-6-deoxy-6-demethyl-9-ethoxythiocarbonylthiotetracycline

[0119] A solution of 1.0 mmole of-8-chloro-4-dedimethylamino-6-deoxy-6-demethyl-7-dimethylamino-9-diazoniumtetracycline hydrochloride in 15 ml of water was addedto a solution of 1.15 mmole of potassium ethyl xanthate in 15 ml ofwater. The mixture was stirred at room temperature for one hour. Thesolid that separated was filtered and dried in a vacuum desiccator.

Example 338-Chloro-9-dimethylamino-4-dedimethylamino-7-dimethylamino-6-deoxy-6-demethytetracyclineSulfate

[0120] To a solution of 100 mg. of the 9-amino compound, from example 6,in 10 ml of ethylene glycol monomethyl ether is added 0.05 ml ofconcentrated sulfuric acid, 0.4 ml of acetaldehyde and 100 mg of a 10%palladium on carbon catalyst. The mixture is reduced with hydrogen atatmospheric pressure and room temperature for 20 minutes. The catalystwas filtered and the filtrate was evaporated to dryness under reducedpressure. The residue is dissolved in 5 ml of methanol and this solutionwas added to 100 ml of ether. The product that separated was filteredand dried.

Example 34 N-(4-methylpiperazin-1-yl)methyl-4-dedimethylamino-6-demethyl-6-deoxytetracycline

[0121] An aqueous solution of 58 mg (37%) formaldehyde (0.72 mmol) wasadded to a solution of 203 mg (0.49 mmol) of4-dedimethylamino-6-demethyl-6-deoxytetracycline in 5.0 ml ethyleneglycol dimethyl ether. The mixture was stirred at room temperature for0.5 hours. 56 mg (0.56 mmol) of 1-methylpiperazine was then added andthe resulting mixture was stirred overnight and refluxed for 20 minutes.The mixture was then cooled and a solid product was collected byfiltration. The solid product was then washed with the solvent and driedby vacuum filtration.

Example 35N-(4-methylpiperazin-1-yl)methyl-4-dedimethylamino-6-demethyl-6-deoxy-9-hexanoylaminotetracycline

[0122] An aqueous solution of 49 mg 37% formaldehyde (0.60 mmol) wasadded to a solution of 146 mg (0.30 mmol) of4-dedimethylamino-6-demethyl-6-deoxy-9-hexanoylaminotetracycline in 5.0ml ethylene glycol dimethyl ether. The mixture was stirred at roomtemperature for 0.5 hours. 60 mg (0.60 mmol) of 1-methylpiperazine wasthen added and the resulting mixture was stirred overnight and refluxedfor 20 minutes. The mixture was then cooled and a solid product wascollected by filtration. The solid product was then washed with thesolvent and dried by vacuum filtration.

Example 364-Dedimethylamino-6-demethyl-6-deoxy-9-hexanoylaminotetracycline

[0123] 1.54 g (7.2 mmol) of hexanoic anhydride and 150 mg of 10% Pd/Ccatalyst were added to 300 mg (0.72 mmol) of4-dedimethylamino-6-demethyl-6-deoxytetracycline in 6.0 ml of1,4-dioxane and 6.0 ml of methanol. The mixture was hydrogenatedovernight at room temperature. The catalyst was removed by filtrationand the filtrate was concentrated under reduced pressure. The residuewas dissolved in 7 ml of ethyl acetate and trituated with 50 ml ofhexane to produce a solid product. The solid product was filtered anddried by vacuum filtration.

Example 37 Phototoxicity Determination

[0124] BALB/c 3T3 (CCL-163) cells were obtained from ATCC and culturedin antibiotic-free Dulbecco's Minimum Essential Medium (4.5 g/lglucose)(DMEM) supplemented with L-glutamine (4 mM) and 10% newborn calfserum. The working cell bank was prepared and found to be free ofmycoplasma. Streptomycin sulfate (100 μg/ml) and penicillin (100 IU/ml)were added to the medium after the cells were treated with test articlein 96-well plates.

[0125] Serial dilutions of the tetracycline derivatives were prepared inDMSO at concentrations 100× to final testing concentration. The CMTdilutions in DMSO were then diluted in Hanks' Balanced Salt Solution(HBSS) for application to the cells. The final DMSO concentration was 1%in treated and control cultures. For the dose range finding assay, 8serial dilutions covered a range of 100 to 0.03 mg/ml in half log stepswhile the definitive assays used 6 to 8 doses prepared in quarter logsteps, centered on the expected 50% toxicity point. In many cases, thedose range for treatment without UV light was different from the doserange selected with UV light. One hundred μg/ml is the highest doserecommended to prevent false negative results from UV absorption by thedosing solutions.

[0126] Controls: Each assay included both negative (solvent) andpositive controls. Twelve wells of negative control cultures were usedon each 96-well plate. Chlorpromazine (Sigma) was used as the positivecontrol and was prepared and dosed like the test tetracyclinederivatives.

[0127] Solar Simulator: A Dermalight SOL 3 solar simulator, equippedwith a UVA H1 filter (320-400 nm), was adjusted to the appropriateheight. Measurement of energy through the lid of a 96-well microtiterplate was carried out using a calibrated UV radiometer UVA sensor.Simulator height was adjusted to deliver 1.7±0.1 m/Wcm² of UVA energy(resulting dose was 1 J/cm² per 10 min.)

[0128] Phototoxicity Assay: Duplicate plates were prepared for each testmaterial by seeding 10⁴ 3T3 cells per well in μl of complete medium 24hours before treatment. Prior to treatment, the medium was removed, andthe cells washed once with 125 μl prewarmed HBSS. Fifty μl of prewarmedHBSS were added to each well. Fifty μl of test article dilutions wereadded to the appropriate wells and the plates returned to the incubatorfor approximately one hour. Following the 1 hr incubation, the platesdesignated for the photoirritation assay were exposed (with the lid on)to 1.7±0.1 mW/cm² UVA light for 50±2 minutes at room temperatureresulting in an irradiation dose of 5J/cm2. Duplicate plates designatedfor the cytotoxicity assay were kept in the dark room temperature for50±2 minutes. After the 50 minute exposure period the test articledilutions were decanted from the plates and the cells washed once with125 μl HBSS. One hundred μl of medium were added to all wells and thecells incubated as above for 24±1 hours.

[0129] After 24 hours of incubation, the medium was decanted and 100 μlof the Neutral Red containing media added to each well. The plates werereturned to the incubator and incubated for approximately 3 hours. After3 hours, the medium was decanted and each well rinsed once with 250 μlof HBSS. The plates were blotted to remove the HBSS and 100 μl ofNeutral Red Solvent were added to each well. After a minimum of 20minutes of incubation at room temperature (with shaking), the absorbanceat 550 m was measured with a plate reader, using the mean of the blankouter wells as the reference. Relative survival was obtained bycomparing the amount of neutral red taken by test article and positivecontrol treated groups to the neutral red taken up by the negative groupon the same plate. IC₅₀ values for both the UVA exposed and non-exposedgroups were determined whenever possible. One dose range finding and atleast two definitive trails were performed on each tetracyclinederivative and control compound.

[0130] Determination of Phototoxicity: Phototoxicity of the tetracyclinederivatives can be measured by its photoirritancy factor (PIF). The PIFwas determined by comparing the IC₅₀ without UVA [IC₅₀(−UVA)] with theIC₅₀ with UVA [IC₅₀(+UVA)]:${PIF} = \frac{{IC}_{50}\left( {- {UVA}} \right)}{{IC}_{50}\left( {+ {UVA}} \right)}$

[0131] If both IC₅₀ values can be determined, the cut off value of thefactor to discriminate between phototoxicants and non-phototoxicants isa factor of 5. A factor greater than 5 is indicative of phototoxicpotential of the test material.

[0132] If IC₅₀ (+UVA) can be determined but IC₅₀(−UVA) cannot, the PIFcannot be calculated, although the compound tested may have some levelof phototoxic potential. In this case, a “>PIF” can be calculated andthe highest testable dose (−UVA) will be used for calculation of the“>PIF.”${\text{>}{PIF}} = \frac{{maximum}\quad {dose}\quad \left( {- {UVA}} \right)}{{IC}_{50}\left( {+ {UVA}} \right)}$

[0133] If both, IC₅₀(−UVA) and IC₅₀(+UVA) cannot be calculated becausethe chemical does not show cytotoxicty (50% reduction in viability) upto the highest dose tested, this would indicate a lack of phototoxicpotential.

Example 38 Effects of Doxycycline Hyclate 20 mg (Dermastat) TabletsAdministered Twice Daily for the Treatment of Moderate Acne

[0134] Study Design:

[0135] Multi-center, randomized double-blind, placebo-controlled;

[0136] Sixty patients enrolled (30 doxycycline and 30 placebo);

[0137] Six month duration of the study.

[0138] Patients received medication twice daily, approximately 12 hoursapart (placebo and drug are identical in appearance.)

[0139] Inclusion Criteria:

[0140] Healthy post-pubescent males and females (age ≧18) with moderatefacial acne:

[0141] Comedones 6 to 200;

[0142] Inflammatory lesions 10 to 75 (papules and pustules, less than orequal to 5 nodules);

[0143] Females tested negative for pregnancy and were non-lactating;

[0144] Females maintained appropriate birth control;

[0145] Patients signed an Informed Consent Form;

[0146] No Accutane treatment for 6 months prior to baseline.

[0147] Exclusion Criteria:

[0148] Use of hormonal contraception 6 months prior to baseline orduring study;

[0149] Use of topical acne treatments within 6 weeks of baseline orduring study;

[0150] Use of systemic antibiotics within 6 weeks of baseline or duringstudy;

[0151] Use of investigational drugs within 90 days of baseline;

[0152] Use of any acne treatments during study.

[0153] Study Procedure:

[0154] Patients reported to clinician at baseline and months 2, 4, and6;

[0155] Acne counts were taken at baseline and months 2, 4, and 6;

[0156] Patient self-assessment and clinician's assessment (baseline, 2,4, and 6);

[0157] Facial photographs at baseline and months 2, 4, and 6;

[0158] Drug dispensation at baseline and months 2 and 4;

[0159] Adverse event recording at baseline and months 2, 4, and 6;

[0160] Microbiological sampling at baseline and month 6;

[0161] Clinical Labs at baseline and month 6.

[0162] Evaluations:

[0163] Efficacy:

[0164] Change in lesion count of papules and pustules

[0165] Change in comedone count

[0166] Change in total lesion count (comedones and inflammatory lesions)

[0167] Microbiology:

[0168] Reduction in skin flora between groups

[0169] Increase in resistant counts between groups

[0170] Efficacy Results

[0171] A six-month treatment with Dermastat resulted in: i) a 53.6%reduction in comedones vis-à-vis a 10.6% reduction of comedones inplacebo (p<0.05); ii) a 50.1% reduction in inflammatory lesionsvis-à-vis a 30.2% reduction of inflammatory lesions in placebo (p<0.01);and iii) a 52.3% reduction in total lesion count vis-à-vis a 17.5%reduction of inflammatory lesions in placebo (p<0.05).

[0172] Microbiology Results:

[0173] A six-month treatment with Dermastat resulted in no reduction ofskin microflora (including Propionibacterium acnes) nor an increase inresistance counts when compared with placebo.

[0174] Thus, while there have been described what are presently believedto be the preferred embodiments of the present invention, those skilledin the art will realize that other and further embodiments can be madewithout departing from the spirit of the invention, and it is intendedto include all such further modifications and changes as come within thetrue scope of the claims set forth herein.

Index of Structures

[0175]

[0176] wherein R7 is selected from the group consisting of hydrogen,amino, nitro, mono(lower alkyl) amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl)amino, halogen, diazonium, di(lower alkyl)amino and RCH(NH₂)CO; Ris hydrogen or lower alkyl; and pharmaceutically acceptable andunacceptable salts thereof; with the following provisos: when either R7and R9 are hydrogen then R8 must be halogen; and when R6-a, R6, R5 andR9 are all hydrogen and R7 is hydrogen, amino, nitro, halogen,dimethylamino or diethylamino, then R8 must be halogen; and when R6-a ismethyl, R6 and R9 are both hydrogen, R5 is hydroxyl and R7 is hydrogen,amino, nitro, halogen or diethylamino, then R8 is halogen; and when R6-ais methyl, R6 is hydroxyl, R5, R7 and R9 are all hydrogen, then R8 mustbe halogen; and when R6-a, R6 and R5 are all hydrogen, R9 is methylaminoand R7 is dimethylamino, then R8 must be halogen; and when R6-a ismethyl, R6 is hydrogen, R5 is hydroxyl, R9 is methylamino and R7 isdimethylamino, then R8 must be halogen; and when R6-a is methyl, R6, R5and R9 are all hydrogen and R7 is cyano, then R8 must be halogen.

[0177] wherein R7 is selected from the group consisting of hydrogen,amino, nitro, mono(lower alkyl) amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R4 is selected from the group consisting of NOH, N—NH-A, andNH-A, where A is a lower alkyl group; R8 is selected from the groupconsisting of hydrogen and halogen; R9 is selected from the groupconsisting of hydrogen, amino, azido, nitro, acylamino, hydroxy,ethoxythiocarbonylthio, mono(lower alkyl) amino, halogen, di(loweralkyl)amino and RCH(NH₂)CO; R is hydrogen or lower alkyl; andpharmaceutically acceptable and unacceptable salts thereof; with thefollowing provisos: when R4 is NOH, N—NH-alkyl or NH-alkyl and R7, R6-a,R6, R5, and R9 are all hydrogen, then R8 must be halogen; and when R4 isNOH, R6-a is methyl, R6 is hydrogen or hydroxyl, R7 is halogen, R5 andR9 are both hydrogen, then R8 must be halogen; and when R4 isN—NH-alkyl, R6-a is methyl, R6 is hydroxyl and R7, R5, R9 are allhydrogen, then R8 must be halogen; and when R4 is NH-alkyl, R6-a, R6, R5and R9 are all hydrogen, R7 is hydrogen, amino, mono(lower alkyl)amino,halogen, di(lower alkyl)amino or hydroxyl, then R8 must be halogen; andwhen R4 is NH-alkyl, R6-a is methyl, R6 and R9 are both hydrogen, R5 ishydroxyl, and R7 is mono(lower alkyl)amino or di(lower alkyl)amino, thenR8 must be halogen; and when R4 is NH-alkyl, R6-a is methyl, R6 ishydroxy or hydrogen and R7, R5, and R9 are all be hydrogen, then R8 mustbe halogen.

[0178] wherein R7, R8, and R9 taken together in each case, have thefollowing meanings: R7 R8 R9 azido hydrogen hydrogen dimethylaminohydrogen azido hydrogen hydrogen amino hydrogen hydrogen azido hydrogenhydrogen nitro dimethylamino hydrogen amino acylamino hydrogen hydrogenhydrogen hydrogen acylamino amino hydrogen nitro hydrogen hydrogen(N,N-dimethyl)glycylamino amino hydrogen amino hydrogen hydrogenethoxythiocarbonylthio dimethylamino hydrogen acylamino dimethylaminohydrogen diazonium dimethylamino chloro amino hydrogen chloro aminoamino chloro amino acylamino chloro acylamino amino chloro hydrogenacylammo chloro hydrogen monoalkylamino chloro amino nitro chloro aminodimethylamino chloro acylamino dimethylamino chloro dimethylaminodimethylamino hydrogen hydrogen hydrogen hydrogen dimethylamino andGeneral Formula (II)

Structure L

Structure M

Structure N

Structure O

[0179] wherein R7, R8, and R9 taken together in each case, have thefollowing meanings: R7 R8 R9 azido hydrogen hydrogen dimethylaminohydrogen azido hydrogen hydrogen amino hydrogen hydrogen azido hydrogenhydrogen nitro dimethylamino hydrogen amino acylamino hydrogen hydrogenhydrogen hydrogen acylamino amino hydrogen nitro hydrogen hydrogen(N,N-dimethyl)glycylamino amino hydrogen amino hydrogen hydrogenethoxythiocarbonylthio dimethylamino hydrogen acylamino hydrogenhydrogen diazonium hydrogen hydrogen dimethylamino diazonium hydrogenhydrogen ethoxythiocarbonylthio hydrogen hydrogen dimethylamino chloroamino amino chloro amino acylamino chloro acylamino hydrogen chloroamino amino chloro hydrogen acylamino chloro hydrogen monoalkyl aminochloro amino nitro chloro amino and General Formula (III)

Structure P

[0180] wherein R8 is hydrogen or halogen and R9 is selected from thegroup consisting of nitro, (N,N-dimethyl)glycylamino, andethoxythiocarbonylthio; and

[0181] wherein R7, R8, and R9 taken together in each case, have thefollowing meanings: R7 R8 R9 amino hydrogen hydrogen nitro hydrogenhydrogen azido hydrogen hydrogen dimethylamino hydrogen azido hydrogenhydrogen amino hydrogen hydrogen azido hydrogen hydrogen nitro bromohydrogen hydrogen dimethylamino hydrogen amino acylamino hydrogenhydrogen hydrogen hydrogen acylamino amino hydrogen nitro hydrogenhydrogen (N,N-dimethyl)glycylamino amino hydrogen amino diethylaminohydrogen hydrogen hydrogen hydrogen ethoxythiocarbonylthio dimethylaminohydrogen methylamino dimethylamino hydrogen acylamino dimethylaminochloro amino amino chloro amino acylamino chloro acylamino hydrogenchloro amino amino chloro hydrogen acylamino chloro hydrogenmonoalkylamino chloro amino nitro chloro amino

[0182] and pharmaceutically acceptable and unacceptable salts thereof.

[0183] wherein R7 is selected from the group consisting of hydrogen,amino, nitro, mono(lower alkyl) amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl) amino, halogen, diazonium, di(lower alkyl)amino and RCH(NH₂)CO; Ris hydrogen or lower alkyl; R^(a) and R^(b) are selected from the groupconsisting of hydrogen, methyl, ethyl, n-propyl and 1-methylethyl withthe proviso that R^(a) and R^(b) cannot both be hydrogen; R^(c) andR^(d) are, independently (CH₂)_(n)CHR^(e) wherein n is 0 or 1 and R^(e)is selected from the group consisting of hydrogen, alkyl, hydroxy,lower(C₁-C₃) alkoxy, amino, or nitro; and, W is selected from the groupconsisting of (CHR^(e))_(m) wherein m is 0-3 and R^(e) is as above, NH,N(C₁-C₃) straight chained or branched alkyl, O, S and N(C₁-C₄) straightchain or branched alkoxy; and pharmaceutically acceptable andunacceptable salts thereof. In a further embodiment, the followingprovisos apply: when either R7 and R9 are hydrogen then R8 must behalogen; and when R6-a, R6, R5 and R9 are all hydrogen and R7 ishydrogen, amino, nitro, halogen, dimethylamino or diethylamino, then R8must be halogen; and when R6-a is methyl, R6 and R9 are both hydrogen,R5 is hydroxyl, and R7 is hydrogen, amino, nitro, halogen ordiethylamino, then R8 is halogen; and when R6-a is methyl, R6 ishydroxyl, R5, R7 and R9 are all hydrogen, then R8 must be halogen; andwhen R6-a, R6 and R5 are all hydrogen, R9 is methylamino and R7 isdimethylamino, then R8 must be halogen; and when R6-a is methyl, R6 ishydrogen, R5 is hydroxyl, R9 is methylamino and R7 is dimethylamino,then R8 must be halogen; and when R6-a is methyl, R6, R5 and R9 are allhydrogen and R7 is cyano, then R8 must be halogen.

Structure K

[0184] wherein: R7, R8, and R9 taken together in each case, have thefollowing meanings: R7 R8 R9 hydrogen hydrogen amino hydrogen hydrogenpalmitamide and STRUCTURE STRUCTURE STRUCTURE STRUCTURE L M N O

[0185] wherein: R7, R8, and R9 taken together in each case, have thefollowing meanings: R7 R8 R9 hydrogen hydrogen acetamido hydrogenhydrogen dimethylaminoacetamido hydrogen hydrogen nitro hydrogenhydrogen amino and STRUCTURE P

[0186] wherein: R8, and R9 taken together are, respectively, hydrogenand nitro.

Structure K

[0187] wherein: R7, R8, and R9 taken together are, respectively,hydrogen, hydrogen and dimethylamino. STRUCTURE STRUCTURE STRUCTURESTRUCTURE C D E F

[0188] wherein R7 is selected from the group consisting of an aryl,alkenyl and alkynyl; R6-a is selected from the group consisting ofhydrogen and methyl; R6 and R5 are selected from the group consisting ofhydrogen and hydroxyl; R8 is selected from the group consisting ofhydrogen and halogen; R9 is selected from the group consisting ofhydrogen, amino, azido, nitro, acylamino, hydroxy,ethoxythiocarbonylthio, mono(lower alkyl) amino, halogen, diazonium,di(lower alkyl)amino and RCH(NH₂)CO; and pharmaceutically acceptable andunacceptable salts thereof; or STRUCTURE STRUCTURE STRUCTURE STRUCTURE CD E F

[0189] wherein: R7 is selected from the group consisting of hydrogen,amino, nitro, mono(lower alkyl) amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of an aryl, alkenyland alkynyl; and pharmaceutically acceptable and unacceptable saltsthereof; or STRUCTURE STRUCTURE STUCTURE STRUCTURE C D E F

[0190] wherein: R7 and R9 are selected from the group consisting of anaryl, alkene, alkyne, or mixures thereof; R6-a is selected from thegroup consisting of hydrogen and methyl; R6 and R5 are selected from thegroup consisting of hydrogen and hydroxyl; R8 is selected from the groupconsisting of hydrogen and halogen; and pharmaceutically acceptable andunacceptable salts thereof. STRUCTURE STRUCTURE STRUCTURE STRUCTURE G HI J

[0191] wherein R7 is selected from the group consisting of an aryl,alkenyl and alkynyl; R6-a is selected from the group consisting ofhydrogen and methyl; R6 and R5 are selected from the group consisting ofhydrogen and hydroxyl; R4 is selected from the group consisting of NOH,N—NH-A, and NH-A, where A is a lower alkyl group; R8 is selected fromthe group consisting of hydrogen and halogen; R9 is selected from thegroup consisting of hydrogen, amino, azido, nitro, acylamino, hydroxy,ethoxythiocarbonylthio, mono(lower alkyl) amino, halogen, di(loweralkyl)amino and RCH(NH₂)CO; and pharmaceutically acceptable andunacceptable salts thereof; or STRUCTURE STRUCTURE STRUCTURE STRUCTURE GH I J

[0192] wherein R7 is selected from the group consisting of hydrogen,amino, nitro, mono(lower alkyl) amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R4 is selected from the group consisting of NOH, N—NH-A, andNH-A, where A is a lower alkyl group; R8 is selected from the groupconsisting of hydrogen and halogen; R9 is selected from the groupconsisting of an aryl, alkenyl and alkynyl; and pharmaceuticallyacceptable and unacceptable salts thereof; or STRUCTURE STRUCTURESTRUCTURE STRUCTURE G H I J

[0193] wherein: R7 and R9 are selected from the group consisting of anaryl, alkenyl, alkynyl; or mixtures thereof; R6-a is selected from thegroup consisting of hydrogen and methyl; R6 and R5 are selected from thegroup consisting of hydrogen and hydroxyl; R4 is selected from the groupconsisting of NOH, N—NH-A, and NH-A, where A is a lower alkyl group; andR8 is selected from the group consisting of hydrogen and halogen; andpharmaceutically acceptable and unacceptable salts thereof.

Structure K

[0194] wherein R7 is selected from the group consisting of an aryl,alkenyl and alkynyl; R8 is selected from the group consisting ofhydrogen and halogen; R9 is selected from the group consisting ofhydrogen, amino, azido, nitro, acylamino, hydroxy,ethoxythiocarbonylthio, mono(lower alkyl) amino, halogen, di(loweralkyl)amino and RCH(NH₂)CO; and pharmaceutically acceptable andunacceptable salts thereof; or

Structure K

[0195] wherein: R7 is selected from the group consisting of hydrogen,amino, nitro, mono(lower alkyl) amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of an aryl, alkenyland alkynyl; and pharmaceutically acceptable and unacceptable saltsthereof; or

Structure K

[0196] wherein: R7 and R9 are selected from the group consisting of anaryl, alkenyl, alkynyl and mixtures thereof; and R8 is selected from thegroup consisting of hydrogen and halogen; and pharmaceuticallyacceptable and unacceptable salts thereof; and STRUCTURE STRUCTURESTRUCTURE STRUCTURE L M N O

[0197] wherein: R7 is selected from the group consisting of an aryl,alkenyl and alkynyl; R8 is selected from the group consisting ofhydrogen and halogen; and pharmaceutically acceptable and unacceptablesalts thereof; or STRUCTURE STRUCTURE STRUCTURE STRUCTURE L M N O

[0198] wherein R7 is selected from the group consisting of hydrogen,amino, nitro, mono(lower alkyl) amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of an aryl, alkenyland alkynyl; and pharmaceutically acceptable and unacceptable saltsthereof; or STRUCTURE STRUCTURE STRUCTURE STRUCTURE L M N O

[0199] wherein R7 is and R9 are selected from the group consisting of anaryl, alkenyl, alkynyl and mixtures thereof; R8 is selected from thegroup consisting of hydrogen and halogen; R9 is selected from the groupconsisting of hydrogen, amino, azido, nitro, acylamino, hydroxy,ethoxythiocarbonylthio, mono(lower alkyl) amino, halogen, di(loweralkyl)amino and RCH(NH₂)CO; and pharmaceutically acceptable andunacceptable salts thereof; and

Structure P

[0200] wherein R9 is selected from the group consisting of an aryl,alkenyl and alkynyl; and R8 is selected from the group consisting ofhydrogen and halogen; and pharmaceutically acceptable and unacceptablesalts thereof; and STRUCTURE Q STRUCTURE R

[0201] wherein R7 is selected from the group consisting of an aryl,alkenyl and alkynyl; R8 is selected from the group consisting ofhydrogen and halogen; R9 is selected from the group consisting ofhydrogen, amino, azido, nitro, acylamino, hydroxy,ethoxythiocarbonylthio, mono(lower alkyl) amino, halogen, di(loweralkyl)amino and RCH(NH₂)CO; and pharmaceutically acceptable andunacceptable salts thereof; or STRUCTURE Q STRUCTURE R

[0202] wherein R7 is selected from the group consisting of hydrogen,amino, nitro, mono(lower alkyl) amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of an aryl, alkenyland alkynyl; and pharmaceutically acceptable and unacceptable saltsthereof; or STRUCTURE Q STRUCTURE R

[0203] wherein R7 and R9 are selected from the group consisting of anaryl, alkenyl, alkynyl; and mixtures thereof, R8 is selected from thegroup consisting of hydrogen and halogen; and pharmaceuticallyacceptable and unacceptable salts thereof.

Structures S-Z

[0204] wherein R7 is selected from the group consisting of an aryl,alkenyl and alkynyl; R6-a is selected from the group consisting ofhydrogen and methyl; R6 and R5 are selected from the group consisting ofhydrogen and hydroxyl; R8 is selected from the group consisting ofhydrogen and halogen; R9 is selected from the group consisting ofhydrogen, amino, azido, nitro, acylamino, hydroxy,ethoxythiocarbonylthio, mono(lower alkyl) amino, halogen, diazonium,di(lower alkyl)amino and RCH(NH₂)CO; R^(a) and R^(b) are selected fromthe group consisting of hydrogen, methyl, ethyl, n-propyl and1-methylethyl with the proviso that R^(a) and R^(b) cannot both behydrogen; R^(c) and R^(d) are, independently, (CH₂)_(n)CHR^(e) wherein nis 0 or 1 and R^(e) is selected from the group consisting of hydrogen,alkyl, hydroxy, lower(C₁-C₃) alkoxy, amino, or nitro; and, W is selectedfrom the group consisting of (CHR^(e))_(m) wherein m is 0-3 and saidR^(e) is as above, NH, N(C₁-C₃) straight chained or branched alkyl, O, Sand N(C₁-C₄) straight chain or branched alkoxy; and pharmaceuticallyacceptable and unacceptable salts thereof; or

Structures S-Z

[0205] wherein R7 is selected from the group consisting of hydrogen,amino, nitro, mono(lower alkyl) amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of an aryl, alkenyland alkynyl; R^(a) and R^(b) are selected from the group consisting ofhydrogen, methyl, ethyl, n-propyl and 1-methylethyl with the provisothat R^(a) and R^(b) cannot both be hydrogen; R^(c) and R^(d) are,independently, (CH₂)_(n)CHR^(e) wherein n is 0 or 1 and R^(e) isselected from the group consisting of hydrogen, alkyl, hydroxy,lower(C₁-C₃) alkoxy, amino, or nitro; and, W is selected from the groupconsisting of (CHR^(e))_(m) wherein m is 0-3 and said R^(e) is as above,NH, N(C₁-C₃) straight chained or branched alkyl, O, S and N(C₁-C₄)straight chain or branched alkoxy; and pharmaceutically acceptable andunacceptable salts thereof; or

Structures S-Z

[0206] wherein: R7 and R9 are selected from the group consisting of anaryl, alkenyl, alkynyl and mixtures thereof; R6-a is selected from thegroup consisting of hydrogen and methyl; R6 and R5 are selected from thegroup consisting of hydrogen and hydroxyl; R8 is selected from the groupconsisting of hydrogen and halogen; R^(a) and R^(b) are selected fromthe group consisting of hydrogen, methyl, ethyl, n-propyl and1-methylethyl with the proviso that R^(a) and R^(b) cannot both behydrogen; R^(c) and R^(d) are, independently, (CH₂)_(n)CHR^(e) wherein nis 0 or 1 and R^(e) is selected from the group consisting of hydrogen,alkyl, hydroxy, lower(C₁-C₃) alkoxy, amino, or nitro; and W is selectedfrom the group consisting of (CHR^(e))_(m) wherein m is 0-3 and saidR^(e) is as above, NH, N(C₁-C₃) straight chained or branched alkyl, O, Sand N(C₁-C₄) straight chain or branched alkoxy; and pharmaceuticallyacceptable and unacceptable salts thereof.

What is claimed is:
 1. A method of treating acne in a human in needthereof comprising administering systemically to said human atetracycline compound in an amount that is effective to treat acne buthas substantially no antibiotic activity, without administering abisphosphonate compound.
 2. A method according to claim 1, wherein saidacne is acne vulgaris, cystic acne, acne atrophica, bromide acne,chlorine acne, acne conglobata, acne cosmetica, acne detergicans,epidemic acne, acne estivalis, acne fulminans, halogen acne, acneindurata, iodide acne, acne keloid, acne mechanica, acne papulosa,pomade acne, premenstral acne, acne pustulosa, acne rosacea, acnescorbutica, acne scrofulosorum, acne urticata, acne varioliformis, acnevenenata, propionic acne, acne excoriee, gram negative acne, steroidacne, or nodulocystic acne.
 3. A method according to claim 1, whereinsaid tetracycline compound is an antibiotic tetracycline compoundadministered in an amount which is 10-80% of the antibiotic amount.
 4. Amethod according to claim 1, wherein said tetracycline compound isdoxycycline administered twice a day in a dose of 20 mg.
 5. A methodaccording to claim 1, wherein said tetracycline compound is minocyclineadministered once a day in a dose of 38 mg.
 6. A method according toclaim 1, wherein said tetracycline compound is minocycline administeredtwice a day in a dose of 38 mg.
 7. A method according to claim 1,wherein said tetracycline compound is minocycline administered threetimes a day in a dose of 38 mg.
 8. A method according to claim 1,wherein said tetracycline compound is minocycline administered fourtimes a day in a dose of 38 mg.
 9. A method according to claim 1,wherein said tetracycline compound is tetracycline administered once aday in a dose of 60 mg/day.
 10. A method according to claim 1, whereinsaid tetracycline compound is tetracycline administered twice a day in adose of 60 mg/day.
 11. A method according to claim 1, wherein saidtetracycline compound is tetracycline administered three times a day ina dose of 60 mg/day.
 12. A method according to claim 1, wherein saidtetracycline compound is tetracycline administered four times a day in adose of 60 mg/day.
 13. A method according to claim 1, wherein saidtetracycline compound is an antibiotic tetracycline compoundadministered in an amount which results in a serum concentration whichis 10-80% of the minimum antibiotic serum concentration.
 14. A methodaccording to claim 1, wherein said tetracycline compound is doxycyclineadministered in an amount which results in a serum concentration whichis 1.0 μg/ml.
 15. A method according to claim 1, wherein saidtetracycline compound is minocycline administered in an amount whichresults in a serum concentration which is 0.8 μg/ml.
 16. A methodaccording to claim 1, wherein said tetracycline compound is tetracyclineadministered in an amount which results in a serum concentration whichis 0.5 μg/ml.
 17. A method according to claim 3 or 13, wherein saidantibiotic tetracycline compound is doxycycline, minocycline,tetracycline, oxytetracycline, chlortetracycline, demeclocycline orpharmaceutically acceptable salts thereof.
 18. A method according toclaim 17, wherein said antibiotic tetracycline compound is doxycycline.19. A method according to claim 18, wherein said doxycycline isadministered in an amount which provides a serum concentration in therange of about 0.1 to about 0.8 μg/ml.
 20. A method according to claim18, wherein said doxycycline is administered in an amount of 20milligrams twice daily.
 21. A method according to claim 19, wherein saiddoxycycline is administered by sustained release over a 24 hour period.22. A method according to claim 21, where said doxcycline isadministered in an amount of 40 milligrams.
 23. A method according toclaim 1, wherein said tetracycline compound is a non-antibiotictetracycline compound.
 24. A method according to claim 23, wherein saidnon-antibiotic tetracycline compound is: 4-de(dimethylamino)tetracycline(CMT-1), tetracyclinonitrile (CMT-2),6-demethyl-6-deoxy-4-de(dimethylamino)tetracycline (CMT-3),4-de(dimethylamino)-7-chlorotetracycline (CMT-4), tetracycline pyrazole(CMT-5) 4-hydroxy-4-de(dimethylamino)tetracycline (CMT-6),4-de(dimethylamino)-12α-deoxytetracycline (CMT-7),6-α-deoxy-5-hydroxy-4-de(dimethylamino)tetracycline (CMT-8),4-de(dimethylamino)-12α-deoxyanhydrotetracycline (CMT-9), or4-de(dimethylamino)minocycline (CMT-10).
 25. A method according to claim23, wherein the non-antibiotic tetracycline compound is selected fromthe group consisting of:

wherein: R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl) amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl) amino, halogen, diazonium, di(lower alkyl)amino and RCH(NH₂)CO; Ris hydrogen or lower alkyl; and pharmaceutically acceptable saltsthereof; with the following provisos: when either R7 and R9 are hydrogenthen R8 must be halogen; and when R6-a, R6, R5 and R9 are all hydrogenand R7 is hydrogen, amino, nitro, halogen, dimethylamino ordiethylamino, then R8 must be halogen; and when R6-a is methyl, R6 andR9 are both hydrogen, R5 is hydroxyl, and R7 is hydrogen, amino, nitro,halogen or diethylamino, then R8 is halogen; and when R6-a is methyl, R6is hydroxyl, R5, R7 and R9 are all hydrogen, then R8 must be halogen;and when R6-a, R6 and R5 are all hydrogen, R9 is methylamino and R7 isdimethylamino, then R8 must be halogen; and when R6-a is methyl, R6 ishydrogen, R5 is hydroxyl, R9 is methylamino and R7 is dimethylamino,then R8 must be halogen; and when R6-a is methyl, R6, R5 and R9 are allhydrogen and R7 is cyano, then R8 must be halogen.
 26. A methodaccording to claim 23, wherein the non-antibiotic tetracycline compoundis selected from the group consisting of:

wherein: R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl)amino, halogen, and di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R4 is selected from the group consisting of NOH, N—NH-A, andNH-A, where A is a lower alkyl group; R8 is selected from the groupconsisting of hydrogen and halogen; R9 is selected from the groupconsisting of hydrogen, amino, azido, nitro, acylamino, hydroxy,ethoxythiocarbonylthio, mono(lower alkyl) amino, halogen, di(loweralkyl)amino and RCH(NH₂)CO; R is hydrogen or lower alkyl; andpharmaceutically acceptable salts thereof; with the following provisos:when R4 is NOH, N—NH-alkyl or NH-alkyl and R7, R6-a, R6, R5, and R9 areall hydrogen, then R8 must be halogen; and when R4 is NOH, R6-a ismethyl, R6 is hydrogen or hydroxyl, R7 is halogen, R5 and R9 are bothhydrogen, then R8 must be halogen; and when R4 is N—NH-alkyl, R6-a ismethyl, R6 is hydroxyl and R7, R5, R9 are all hydrogen, then R8 must behalogen; and when R4 is NH-alkyl, R6-a, R6, R5 and R9 are all hydrogen,R7 is hydrogen, amino, mono(lower alkyl)amino, halogen, di(loweralkyl)amino or hydroxyl, then R8 must be halogen; and when R4 isNH-alkyl, R6-a is methyl, R6 and R9 are both hydrogen, R5 is hydroxyl,and R7 is mono(lower alkyl)amino or di(lower alkyl)amino, then R8 mustbe halogen; and when R4 is NH-alkyl, R6-a is methyl, R6 is hydroxy orhydrogen and R7, R5, and R9 are all be hydrogen, then R8 must behalogen.
 27. A method according to claim 23 wherein the non-antibiotictetracycline compound is selected from the group consisting of:

wherein: R7, R8, and R9 taken together in each case, have the followingmeanings: R7 R8 R9 azido hydrogen hydrogen dimethylamino hydrogen azidohydrogen hydrogen amino hydrogen hydrogen azido hydrogen hydrogen nitrodimethylamino hydrogen amino acylamino hydrogen hydrogen hydrogenhydrogen acylamino amino hydrogen nitro hydrogen hydrogen(N,N-dimethyl)glycylamino amino hydrogen amino hydrogen hydrogenethoxythiocarbonylthio dimethylamino hydrogen acylamino dimethylaminohydrogen diazonium dimethylamino chloro amino hydrogen chloro aminoamino chloro amino acylamino chloro acylamino amino chloro hydrogenacylamino chloro hydrogen monoalkylamino chloro amino nitro chloro aminodimethylamino chloro acylamino dimethylamino chloro dimethylaminohydrogen hydrogen dimethylamino dimethylamino hydrogen hydrogen and

Structure L

Structure M

Structure N

Structure O

wherein: R7, R8, and R9 taken together in each case, have the followingmeanings: R7 R8 R9 azido hydrogen hydrogen dimethylamino hydrogen azidohydrogen hydrogen amino hydrogen hydrogen azido hydrogen hydrogen nitrodimethylamino hydrogen amino acylamino hydrogen hydrogen hydrogenhydrogen acylamino amino hydrogen nitro hydrogen hydrogen(N,N-dimethyl)glycylamino amino hydrogen amino hydrogen hydrogenethoxythiocarbonylthio dimethylamino hydrogen acylamino hydrogenhydrogen diazonium hydrogen hydrogen dimethylamino diazonium hydrogenhydrogen ethoxythiocarbonylthio hydrogen hydrogen dimethylamino chloroamino amino chloro amino acylamino chloro acylamino hydrogen chloroamino amino chloro hydrogen acylamino chloro hydrogen monoalkylaminochloro amino nitro chloro amino and

Structure P

wherein: R8 is hydrogen or halogen and R9 is selected from the groupconsisting of nitro, (N,N-dimethyl)glycylamino, andethoxythiocarbonylthio; and

wherein: R7, R8, and R9 taken together in each case, have the followingmeanings: R7 R8 R9 amino hydrogen hydrogen nitro hydrogen hydrogen azidohydrogen hydrogen dimethylamino hydrogen azido hydrogen hydrogen aminohydrogen hydrogen azido hydrogen hydrogen nitro bromo hydrogen hydrogendimethylamino hydrogen amino acylamino hydrogen hydrogen hydrogenhydrogen acylamino amino hydrogen nitro hydrogen hydrogen(N,N-dimethyl)glycylamino amino hydrogen amino diethylamino hydrogenhydrogen hydrogen hydrogen ethoxythiocarbonylthio dimethylamino hydrogenmethylamino dimethylamino hydrogen acylamino dimethylamino chloro aminoamino chloro amino acylamino chloro acylamino hydrogen chloro aminoamino chloro hydrogen acylamino chloro hydrogen monoalkylamino chloroamino nitro chloro amino and pharmaceutically acceptable salts thereof.


28. A method according to claim 23, wherein the non-antibiotictetracycline compound is selected from the group consisting of:

wherein: R7 is selected from the group consisting of hydrogen, amino,nitro, mono(lower alkyl) amino, halogen, di(lower alkyl)amino,ethoxythiocarbonylthio, azido, acylamino, diazonium, cyano, andhydroxyl; R6-a is selected from the group consisting of hydrogen andmethyl; R6 and R5 are selected from the group consisting of hydrogen andhydroxyl; R8 is selected from the group consisting of hydrogen andhalogen; R9 is selected from the group consisting of hydrogen, amino,azido, nitro, acylamino, hydroxy, ethoxythiocarbonylthio, mono(loweralkyl) amino, halogen, diazonium, di(lower alkyl)amino and RCH(NH₂)CO; Ris hydrogen or lower alkyl; R^(a) and R^(b) are selected from the groupconsisting of hydrogen, methyl, ethyl, n-propyl and 1-methylethyl withthe proviso that R^(a) and R^(b) cannot both be hydrogen; R^(c) andR^(d) are, independently, (CH₂)_(n)CHR^(e) wherein n is 0 or 1 and R^(e)is selected from the group consisting of hydrogen, alkyl, hydroxy,lower(C₁-C₃)alkoxy, amino, or nitro; and, W is selected from the groupconsisting of (CHR^(e))_(m) wherein m is 0-3 and said R^(e) is as above,NH, N(C₁-C₃) straight chained or branched alkyl, O, S and N(C₁-C₄)straight chain or branched alkoxy; and, pharmaceutically acceptablesalts thereof.
 29. A method according to claim 16, wherein thenon-antibiotic tetracycline compound selected from the group consistingof structures S-Z has the following provisos: when either R7 and R9 arehydrogen then R8 must be halogen; and when R6-a, R6, R5 and R9 are allhydrogen and R7 is hydrogen, amino, nitro, halogen, dimethylamino ordiethylamino, then R8 must be halogen; and when R6-a is methyl, R6 andR9 are both hydrogen, R5 is hydroxyl, and R7 is hydrogen, amino, nitro,halogen or diethylamino, then R8 is halogen; and when R6-a is methyl, R6is hydroxyl, R5, R7 and R9 are all hydrogen, then R8 must be halogen;and when R6-a, R6 and R5 are all hydrogen, R9 is methylamino and R7 isdimethylamino, then R8 Pmust be halogen; and when R6-a is methyl, R6 ishydrogen, R5 is hydroxyl, R9 is methylamino and R7 is dimethylamino,then R8 must be halogen; and when R6-a is methyl, R6, R5 and R9 are allhydrogen and R7 is cyano, then R8 must be halogen.
 30. A methodaccording to claim 1, wherein said tetracycline compound has aphotoirritancy factor of less than the photoirritancy factor ofdoxycycline.
 31. A method according to claim 1, wherein saidtetracycline compound has a photoirritancy factor from about one toabout two.
 32. A method according to claim 31, wherein said tetracyclinecompound has a general formula:

wherein R7, R8, and R9 taken together are, respectively, hydrogen,hydrogen and dimethylamino.
 33. A method according to claim 1, whereinsaid tetracycline compound has a photoirritancy factor from about 1.0 toabout 1.2.
 34. A method according to claim 33, wherein said tetracyclinecompound is selected from the group consisting of:

wherein R7, R8, and R9 taken together in each case, have the followingmeanings: R7 R8 R9 hydrogen hydrogen amino hydrogen hydrogen palmitamideand

Structure L

Structure M

Structure N

Structure O

wherein R7, R8, and R9 taken together in each case, have the followingmeanings: R7 R8 R9 hydrogen hydrogen acetamido hydrogen hydrogendimethylaminoacetamido hydrogen hydrogen nitro hydrogen hydrogen aminoand

Structure P

wherein R8, and R9 taken together are, respectively, hydrogen and nitro.35. A method according to claim 1, wherein said systemic administrationis oral administration, intravenous injection, intramuscular injection,subcutaneous administration, transdermal administration or intranasaladministration.
 36. A method of treating acne in a human in need thereofcomprising administering to said human an effective amount of anon-antibiotic tetracycline compound without administering abisphosphonate compound.
 37. A method according to claim 36, whereinsaid administration is topical administration.
 38. A method according toclaim 36, wherein said administration is systemic administration.
 39. Amethod for reducing the number of comedones in a human in need thereofcomprising administering systemically to said human a tetracyclinecompound in an amount that is effective to reduce the number ofcomedones but has substantially no antibiotic activity.
 40. A methodaccording to claim 39, wherein said tetracycline compound isdoxycycline.
 41. A method according to claim 40, wherein saiddoxycycline is administered in a daily amount of from about 30 to about60 milligrams but maintains a concentration in human plasma below thethreshold for a significant antibiotic effect.
 42. A method according toclaim 40, wherein said doxycycline is administered in an amount ofapproximately 20 milligrams twice daily.
 43. A method according to claim39, wherein said tetracycline compound is administered withoutadministering a bisphosphonate.
 44. A method for inhibiting oxidation ofmelanin in a human in need thereof comprising administering systemicallyto said human a tetracycline compound in an amount that is effective toinhibit oxidation of melanin but has substantially no antibioticactivity.
 45. A method for inhibiting lipid-associated abnormalfollicular differentiation in a human in need thereof comprisingadministering systemically to said. human a tetracycline compound in anamount that is effective to inhibit lipid-associated abnormal folliculardifferentiation but has substantially no antibiotic activity.